This invention relates to novel monomeric analogues of human insulin (HI) obtainable by recombinant DNA technology.
Insulin is highly effective in treating insulin-dependent diabetes, and has been used clinically for nearly 80 years. With advances in DNA technology and the development of biotechnology industries, insulin extracted from animal pancreas is gradually being replaced by recombinant forms of human insulin, produced in microbial systems. This trend is encouraged by two observations, the number suffering from diabetes mellitus is on the increase globally and the clinical dose required to treat them is in milligram (mg) quantities.
Currently, the organisms employed for the commercial production of recombinant human insulin are E. coli and S. cerevisiae. The expression levels in E. coli are high but difficulties associated with downstream purification often lead to loss of yield. These difficulties are not encountered with S. cerevisiae, because the insulin produced is secreted into the culture medium, facilitating purification. However, the level of expression observed in this organism is low and difficult to increase.
Until recently, introduction of Lispro(copyright), clinical preparations of human insulin contained polymeric forms of insulin which are slow acting. Monomeric forms of insulin, as described in U.S Pat. No. 5618913, by contrast, are relatively fast-acting and mimic more closely the natural situation. They therefore demonstrate a great potential for clinical application. A commercial monomeric insulin, available as Lispro(copyright), comprises inversion of amino acids 28 and 29 of the B chain of human insulin, and may be abbreviated as B28Lys,B29Pro.
Kristensen et al, J. Biol. Chem. 272(20):12978-83 (1997), discloses alanine substitution at various positions on the insulin molecule, including B12, B16 and B26. A single substitution with Ala affected the binding activity of the resultant insulin analogue in certain cases.
Wang et al, Biochem. Mol. Biol. Int. 39(6):1245-54 (1996), discloses B12Thr, i.e. an insulin analogue in which the 12th amino acid of the B-chain of human insulin (Val) is substituted by Thr. Again, an effect on binding activity was observed.
EP-A-0046979 discloses des-B30 derivatives of human insulin.
EP-A-0291863 discloses des-B1 derivatives of human insulin.
According to the present invention, novel human insulin analogues are monomeric variants of B12Thr, B16Ala and B26Ala; the latter have not previously been recognised as monomeric. In addition to replacement of any or all of the 12th, 16th and 26th amino acids on the B-chain, such that the analogue is monomeric, the B-1 and/or B-30 terminal amino acids may be absent. The term xe2x80x9cinsulin analoguexe2x80x9d as used herein means a compound having a molecular structure similar to that of human insulin, including disulphide bridges between A7Cys and B7Cys and between A20Cys and B19Cys, and an internal disulphide bridge between A6Cys and A11Cys, and having insulin activity.
Without wishing to be bound by theory, it appears that, in the primary structure of the insulin molecule, a number of the amino acids in the B-chain are responsible for the polymerisation of insulin in clinical preparations. These include those in positions B12, B16 and B26. In particular, the replacement of Val by Thr in position B12 or Tyr by Ala in position B16 or B26 significantly reduces the tendency of the insulin analogues to polymerise even at high concentrations (see Example 9). This enhanced tendency to exist as a monomeric structure is not affected by deletion of either one or both of the terminal amino acids of the B-chain.
The Scheme, below, shows the construction of the expression plasmids pNHI-2/AOX1, pNHI-3/AOX1, pNHI4/AOX1 and the engineering of recombinant cells YP99/NHI-2, YP99/NHI-3 and YP99/NHI-4. It sets out a representative procedure for the preparation of compounds of the invention, by analogy with the use of the human insulin target gene (HI) housed in the shuttle plasmid pHI/PGK. This shuttle vector is constructed from the plasmid pVT102-U (acquired from Canadian Research Institute) and subsequently multiplied by PCR (Maniatis et al (1989), Molecular Cloning A Laboratory Manual, 2nd ed. New York: Cold Spring Harbour Laboratory), to obtain multiple copies of human insulin target gene (HI) and flanking alpha mating factor leader (MFL) sequence. The target gene is then cloned into plasmid pPIC9 which is subsequently linearised with BglII prior to being employed to transform P. pastoris cell GS115 by the spheroplast method. Once plasmid pPIC9 containing the target gene is intemalised, it integrates into the chromosomal DNA of the host cell [1]. Transformed cells bearing a high copy number of the HI gene are selected using the antibiotic G418 by the method described by Scover et al [2]. The presence of multiple copies of the HI are ascertained by the dot blotting method [3]. Cells bearing a high copy number of the HI gene are utilised to generate the human insulin precursor by fermentation, and after purification converted to human insulin by tryptic transpeptidation.
In order to obtain recombinant forms of human insulin analogues according to this invention, target genes were produced. This was accomplished by the xe2x80x9cgap double-stranded DNAxe2x80x9d method described by Li Yiping et al. (1987Biotech. J. 3:90) which permits site-directed mutations in the HI target gene. Primers specifically designed to give B12Thr, B16Ala and B26Ala were as follows; For B12Thr (NHI-2): refer to Wang et al., supra For B16Ala (NHI-3): 5xe2x80x2 TGA GGC TTT GNN STT GGT TTG CG 3xe2x80x2 (SEQ ID No.1) in which N can be any nucleotide (G,A,T or C), and S is C or G. For B26Ala (NHI-4): 5xe2x80x2 GAA AGA GGTT TTC NNS ACT CCT AGG GC 3xe2x80x2 (SEQ ID No.2) in which N and S are as defined above.
Novel human insulin analogues may be obtained by removing B30Thr and/or B1Phe, e.g. yielding a des-B1 and/or des-B30 analogue. Deletion may be achieved by known methodology. Rather than tryptic transpeptidation, to produce des-B30 human insulin, limited hydrolysis has been adopted, using trypsin in the preferred method, which further simplifies the process and increases the yield of insulin.
The methylotrophic yeast, Pichia pastoris is the preferred host for use In this invention for the preparation of insulin analogues because, as the Examples show, it has the advantages of high expression, simple processing, low production cost and high density culture. Furthermore it offers the advantages of a eukaryotic cell system; the correct folding and post-translational processing of secreted protein These advantages greatly enhance the possibility of utilizing P. pastoris as the expression host in the scale-up of human insulin production. Its use in the expression of proteins of commercial importance has been documented elsewhere [3-5].
Human insulin analogues of the invention may be used in therapy. Their application and utility will be readily evident to those of ordinary skill in the art, e.g. in the treatment of diabetes mellitus.